In 2009 approximately 225 million cases of malaria were reported resulting in 781,000 deaths, mostly in children under 5 years old. Of the 5 Plasmodium species that cause malaria, P. falciparum has the highest case fatality rate and is subsequently the most studied. This has resulted in a decrease in the prevalence of P. falciparum, yet in many of the countries where falciparum malaria has been eradicated, vivax malaria remains endemic. P. vivax is the most geographically widespread of the Plasmodium spp., and over one-third of the world's population is at risk of infection. P. vivax is uniquely different from P. falciparum in that P. vivax is able to persist in a dormant, asymptomatic stage in the liver that can reemerge months to years after initial infection to cause clinical disease. These relapse episodes help to sustain transmission in endemic regions. Primaquine is the only licensed drug that will eliminate dormant liver-stage P. vivax parasites and thus prevent relapse. The development of resistance is a consistent hurdle faced when trying to control malaria and primaquine resistance has been reported. Despite being in use for over 50 years, the mechanism of action of primaquine is unknown. The objective of this fellowship application is to identify the molecular determinants of primaquine resistance in Plasmodium spp. Identification of these determinants will inform the development of new antimalarials that target liver-stage P. vivax. To accomplish this objective, I will use an in vivo model to evolve primaquine resistance in Plasmodium parasites. P. vivax cannot be cultured in vitro, therefore I will use a rodent model of malaria, P. berghei. First I will develop a whole-genome sequencing approach to detect genetic polymorphisms in P. berghei. I will then evolve parasite resistance to primaquine by serial passage of blood stages of P. berghei in mice treated with increasing concentrations of primaquine. The whole-genome sequence of the starting evolution strain can then be compared with the resistance-evolved strain to determine genetic modifications that are associated with primaquine resistance. This resistant line can then be tested to see if it is also resistant in the liver stage by allowing mosquitoes infected with the resistant line to feed on mice treated with primaquine and subsequently see if a blood-stage infection develops. Identification of the molecular determinants of primaquine resistance can then be used to establish molecular markers that can detect resistance in field isolates. These molecular determinants will also inform drug discovery efforts to identify additional liver-stage acting antimalarials. Both applications of the proposed results will aide in the eradication of vivax malaria.